• The Korean Journal of Zoology
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• v.35 no.3
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• pp.308-321
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• 1992

심근세포는 골격근 세포와 더불어 세포의 분화기작을 이해하기 위한 좋은 모델이라 할 수 있다. 본 연구에서는 심근세포의 배양중에 일어나는 심근 근원섬유형성과정에 대한 이해를 위하여 SDS- 및 two-dimensional gel electrophoresis와 immunofluorescent 및 immunoelectron microscopy를 수단으로 이를 검토하였다. 배양과정에서 나타나는 주요한 단백질 중에서 SDS-PAGE에 의해 209 kDa와 53 kDa 및 46 kDa band가 각각 막분획 및 세포질 분획 단백질에서 배양과정동안에 가장 현저한 양상을 보였다. 특히 배양 3일과 4일의 양상에서 뚜렷한 변화를 나타내어 이들의 OD 격은 배양 96시간에서의 막분획에서 0.38(209 kDa)와 0.52(53 kDa) 및 배양 72시간에서의 세포질 분획에서 0.34(46 kDa)로 각각 최고치를 나타내었다. 또한 이차원 전기영동상의 양상에서도 세포질 및 막분획 단백질의 전반적인 전개양상이 배양 96시간에서 가장 두드러짐으로써 결국 배양 72시간 및 96시간대에서 심근 모세포의 분화와 관련된 일련의 단백질 합성과정이 가장 활발하다는 것을 알 수 있었다. 배양중에 발현되는 actin에 대한 형광현미경적 분석에 의해, actin은 세포내에서 불균등하게 분포하였고 근원섬유가 형성되는 시기인 배양 96시간에 세포외곽부에서 나타나는 다소 강한 염색성을 관찰할 수 있었다. 심근세포의 근원섬유는 전자현미경 관찰에 의해 primitive forms로 배양 96시간에 출현함을 알 수 있었으며 이 시기의 근원섬유의 형태는 여러 구성대(A, H and M bands)가 미약하나 1대는 비교적 뚜렷하였다. 따라서 심근세포의 T근원섬유형성과정에서 관찰되는 뚜렷한 단백질양상의 변화와 근원섬유의 출현시기 및 구성과정간에는 중요한 관련성이 있으며 이러한 과정에서 actin은 세포질내에서 불균등한 분포를 나타낸다는 것을 알 수 있었다.

• Biomedical Science Letters
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• v.2 no.1
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• pp.1-12
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• 1996

Antigenic components reacting with IgE and IgG antibodies were localized in muscular layer of adult and of larva, sparganum. But the antigenic components inducing IgG were localized at tegument and parenchyma in addition to muscular layer in adult and sparganum. Also in sparganum, the surface of calcareous corpuscles of parenchyma showed immunoreactivity to IgG antibody. However antigenic components inducing IgE antibody were not localized in tegument and parenchyma, but in adult worm, we observed the immunopositive reaction at the lining of vitelline follicles in mature proglottis and on surface of egg shell within uterus of graved proglottis. By the method of immunogold-labelling, we observed the location of antigenic particles in tegument of sparganum. The density of antigenic particles inducing IgG was higher than that of antigen particles inducing IgE in syncytial tegument, tegument cells. A total of 43 and 36 protein bands were resolved from crude extracts of adult and sparganum, respectively, by SDS-PAGE. 34 bands from crude extracts of adult and larva were migrated to same positions. By EITB, 21 bands of 44 bands in adult were recognized with IgG antibody, and also 21 bands of 36 bands in sparganum. 13 bands of them were common antigenic components both in the adult worm and sparganum. Because 19 bands of 44 bands in adult worm were reacted with IgE antibody, they were IgE antigenic component. In sparganum, 13 bands were IgE antigenic components. 9 bands of them were common antigenic component inducing IgE antibody in both a-dult and sparganum. 3 bands of antigenic component recognized by IgE and IgG antibody were nonspecific antigen in both adult and sparganum of Spirometra erinacei.

• Applied Microscopy
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• v.40 no.4
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• pp.261-266
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• 2010

ATP is the energy source synthesized at the electron transferase that consist of complex I, II, III, IV and V in mitochondrial cristae. The complex V functions as ATPase which composed of sub-complex $F_0$ and $F_1$. Porin or VDAC (voltagedependent anion-selective channel), is a family of small pore-forming proteins of the mitochondrial outer membrane, and play important roles in the regulated flux of anion, proton and metabolites between the cytosolic and mitochondrial compartments. The channel allows the diffusion of negatively charged solutes such as succinate, malate, and ATP in the fully open state, but of positively charged ions in subconducting state. In this study, in order to investigate the relationship of the function and localization between porin and ATPase we observed the distribution of porin and ATPase in the mitochondria of the bovine heart. Monoclonal antibodies against porin and ATPase ${\beta}$-subunit were used to detect porin and ATPase using light microscope with immunohistochemistry and immunofluorescence, and using electron microscope with immunogold-labeling. ATPase were stained in longitudinal section region in cardiac muscle, porin were stained in longitudinal section region in cardiac muscle. We viewed more specific pattern of localization and distribution of these proteins using immunofluorescence method. There were some region which were labeled with porin or ATPase respectively, and others which were labeled both proteins in cardiac muscle. The electron microscope results showed that immunogold labeled porin were labeled locally at mitochondrial outer membrane and ATPase were labeled evenly at mitochondrial cristae. But ATPase was not labeled at mitochondria cristae. These results confirmed the subcellular localizations of porin and ATPase in mitochondrial outer membrane and cristae. Also, we assumed that ATP synthesis always does not activation in all mitochondria exist in the bovine cardiac muscle.

• Tuberculosis and Respiratory Diseases
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• v.59 no.4
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• pp.397-405
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• 2005

Background : Laminin-1 is known to have regular functions in the development and course of differentiation of the lungs. The morphogenesis and distribution of laminin-1 still remains as a mystery and its distribution and changes in the molecular structure of laminin-1 in the pathogenesis of the lung still is a subject of great controversy. In this study, experiments were done to delineate the distribution and changes in the amount of laminin-1 after inducing inflammation of the lungs by exposing experimental animals to CS gas and especially, to find compositions of laminin-1 within type II pneumocytes. Materials and Methods : The experimental subjects of study were newborn rats and the extracted tissue from the experimental rats were viewed under light microscope and electron microscope after the sections were treated with immunohistochemical methods and immunogold reaction methods using bounded gold particles. Results : 1) Lymphocytes and mononuclear phagocytes invaded the alveolar septa in the 2 day group rats after CS gas exposure and intense interstitial inflammation was seen in the 3 day group. 2) Laminin immunoreactions decreased to a moderate degree in the 2 and 3 day group rats after CS gas exposure and strong laminin immunoreactions were seen again in the 5 and 7 day group rats. 3) Gold particles in basal lamina of the lung blood-air barrier decreased and in the type I pneumocytes decreased in the 2 and 3 day group rats after CS gas exposure. 4) Gold particles were seen only on the surface of the cell membranes of type II pneumocytes of the 1 and 2 day group rats after CS gas exposure. 5) Few gold particles around the lamellar bodies and cytoplasm of type II pneumocytes in the control rat group and at 12 hours after CS gas exposure. Gold particles are seen only on the surface of type II pneumocytes of the 1 and 2 day group rats after CS gas exposure and are evenly distributed in small amounts in the cells of the 3 day group after CS gas exposure. Conclusion : CS gas exposure in the rats caused inflammation of lung alveolar septa and also induced a decrease in laminin-1 in basal lamina and loss of laminin-1 in the cytoplasm of type II pneumonocytes. As the inflammatory cells disappeared, an increase in the distribution of laminin-1 occurred. This reflects tissue regeneration functions of laminin-1 in the pneumocytes of rats and the distribution of laminin-1 in type II pneumocytes can be seen through the electron microscope using immunogold methods.

• KSBB Journal
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• v.21 no.5
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• pp.325-330
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• 2006

This study presents the characterization of an integrated portable microfluidic electrical detection system for fast and low volume immunoassay using polystyrene microbead, which are used as immobilization surfaces. In our chip, a filtration method using the microbead was adopted for sample immobilization and immunogold silver staining(IGSS) was used to increase the electrical signal. The chip is composed of an inexpensive and biocompatible Polydimethylsiloxane(PDMS) layer and Pyrex glass substrate. Platinum microelectrodes for electric signal detection were fabricated on the substrate and microchannel and pillar-type microfilters were formed in the PDMS layer. With a fabricated chip, we reacted antigen and antibody according to the procedures. Then, silver enhancer was injected to increase the size of nanogold particles tagged with the second antibody. As a result, microbeads were connected to each other and formed an electrical bridge between microelectrodes. Resistance measured through the electrodes showed a difference of two orders of magnitude between specific and nonspecific immuno-reactions. The detection limit was 10 ng/ml. The developed immunoassay chip reduced the total analysis time from 3 hours to 50 min. Fast and low-volume biochemical analysis has been successfully achieved with the developed microfilter and immuno-sensor chip, which is integrated to the microfluidic system.

• Parasites, Hosts and Diseases
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• v.36 no.4
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• pp.227-234
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• 1998

Actin and tropomyosin of Cryptosporidium muris were localized by immunogold labeling. Two kinds of antibodies for actin labeling were used. The polyclonal antibody to skeletal muscle (chicken back muscle) actin was labeled on the pellicle and cytoplasmic vacuoles of parasites. The feeder organelle has showed a small amount of polyclonal actin antibody labeling as well. Whereas the monoclonal antibody to smooth muscle (chicken gizzard muscle) actin was chiefly labeled on the filamentous cytoplasm of parasites. The apical portion of host gastric epithelial cell cytoplasm was also labeled by smooth muscle actin together. The polyclonal antibody to tropomyosin was much more labeled at C. muris than host cells, so it could be easily identified even with low magnification (${\times}2,000$). The tropomyosin was observed along the pellicle, cytoplasmic vacuoles, and around the nucleus also. The skeletal muscle type actin seems to play a role in various celluar functions with tropomyosin in C. muris; on the other hand, the smooth muscle type actin was located mainly on the filamentous cytoplasm and supported the parasites firm attachment to host cells. Tropomyosin on the pellicle was thought to be able to stimulate the host as a major antigen through continuous shedding out by the escape of sporozoites or merozoites from their mother cells.

• Korean Journal of Environmental Biology
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• v.25 no.4
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• pp.289-296
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• 2007

Di-(ethyhexyl) phthalate (DEHP), commonly used as a plasticizer for manufacturing flexible vinyl products, has been the topic of extensive research, especially concerning endocrine disrupting properties. Metallothionein (MT) is a low molecular weight (6,000$\sim$7,000 Da), cysteine-rich (22$\sim$23%), metal-binding protein and is known to be induced by extrinsic factors such as chemical agents and stresses. Some of the known function of MT include detoxification of heavy metals and alkylating agents and neutralization of free radicals. Nonetheless, the definitive physiological function of MT are still unknown. This study was carried out to investigate the effects of DEHP on the ultrastructural changes and the expression of MT of the rat liver. The rats were orally intubated with either corn oil (experimental control) or 0.5 mg, 1.5 mg and 4.5 mg DEHP kg$^{-1}$ day$^{-1}$ in 0.5 mL of corn oil for 15 days before sacrificing and sampling. DEHP induced mild ultrastrctural changes of some cell organelles such as rough endoplasmic reticulum, mitochondria, lysosomes and peroxisomes in the rat liver treated with DEHP. In the respect of immunogold labelling and Western blotting, MT expression of the liver tissue was up-regulated by DEHP. In conclusion, DEHP has effects on the ultrastructures and hepatic function for MT expression in rat.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 1995.06b
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• pp.27-53
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• 1995

In plant pathology there is an increasing necessity for improved cytological techniques as basis for the localization of cellular substances within the dynamic fine structure of the host-(plant)-pathogen-interaction. Low temperature (LT) preparation techniques (shock freezing, freeze substitution, LT embedding) are now successfully applied in plant pathology. They are regarded as important tools to stabilize the dynamic plant-pathogen-interaction as it exists under physiological conditions. - The main advantage of LT techniques versus conventional chemical fixation is seen in the maintenance of the hydration shell of molecules and macromolecular structures. This results in an improved fine structural preservation and in a superior retention of the antigenicity of proteins. - A well defined ultrastructure of small, fungal organisms and large biological samples such as plant material and as well as the plant-pathogen (fungus) infection sites are presented. The mesophyll tissue of Arabidopsis thaliana is characterized by homogeneously structured cytoplasm closely attached to the cell wall. From analyses of the compatible interaction between Erysiphe graminis f. sp. hordei on barley (Hordeum vulgare), various steps in the infection sequence can be identified. Infection sites of powdery mildew on primary leaves of barley are analysed with regard to the fine structural preservation of the haustoria. The presentation s focussed on the ultrastructure of the extrahaustorial matrix and the extrahaustorial membrane. - The integration of improved cellular preservation with a molecular analysis of the infected host cell is achieved by the application of secondary probing techniques, i.e. immunocytochemistry. Recent data on the characterization of freeze substituted powdery mildew and urst infected plant tissue by immunogold methodology are described with special emphasis on the localization of THRGP-like (threonine-hydrxyproline-rich glycoprotein) epitopes. Infection sites of powdery mildew on barley, stem rust as well as leaf rust (Puccinia recondita) on primary leaves of wheat were probed with a polyclonal antiserum to maize THRGP. Cross-reactivity with the anti-THRGP antiserum was observed over the extrahaustorial matrix of the both compatible and incompatible plant-pathogen interactions. The highly localized accumulation of THRGP-like epitopes at the extrahaustorial host-pathogen interface suggests the involvement of structural, interfacial proteins during the infection of monocotyledonous plants by obligate, biotrophic fungi.

• Applied Microscopy
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• v.38 no.2
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• pp.125-133
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• 2008

Cytochrome-c-oxidase in mitochondria membrane is one of the most important factors for energy generation in the cell. As well as it is electron transfer enzyme, it is also heavily related to the apoptosis and other pathologic conditions. Meanwhile, porin is a protein located in inner and outer membranes of mitochondria, which is assumed to be functionally correlated with cytochrome-c-oxidase. It functions as forming electron transfer chain and conveying ATP. Therefore, using the immune-microscopy, It compared the distribution of cytochrome-c-oxidase and porin to figure out the formation and changes on cytochrome-c-oxidase in mitochondrial cristae. The sarcroplasm of cardic muscle tissue has many mitochondria. They are classified into two groups: the mitochondria with many cytochrome-c-oxidase and the mitochondria with only porins. The mitochondria with porins had few cytochrome-c-oxidases in their membrane; in contrast, the other mitochondria with rich cytochrome-c-oxidase had few porins in their walls. In addition, according to the location of the tissue in bovine heart, distribution of those kind of mitochondria had been clearly separated. As a result, it could be assumed that immature mitochondria has many porins to transfer the protein materials from sarcroplasm through the porins, and they made cytochrome-c-oxidase until it is enough, and then they decreased the porin and maintained minimum number of the porin.

• Applied Microscopy
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• v.38 no.4
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• pp.375-382
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• 2008

In order to investigate shape of synaptic vesicles of the tooth pulp afferent boutons and their presynaptic endings (p-endings), and the neuroactive substance of the p-endings in the trigeminal nucleus principalis, rat incisor tooth pulp afferents were labeled by the horseradish peroxidase (HRP) and quantitative ultrastructural analysis and postembedding immunogold labeling were performed. Labeled tooth pulp afferent boutons contained clear, spherical synaptic vesicles (diameter: $45{\sim}55\;nm$) and occasionally dense core vesicles(diameter: $80{\sim}120\;nm$). They formed symmetrical synapses with unlabeled axon terminals (p-endings) containing pleomorphic synaptic vesicles. The ratio of short to long diameter (form factor) of synaptic vesicles of pulp afferent boutons was 0.6 to 0.99, whereas that of p-endings was 0.25 to 0.99. In addition, most of the p-endings showed GABA-like immunoreactivity. These results indicate that the shape of synaptic vesicles is quite different between the tooth pulp afferent boutons and p-endings, and the p-endings may contain GABA as a neuroactive substance in the trigeminal nucleus principalis.